The long-term goal of my research is to understand the factor(s) determining the tumorigenic state of a cell, and the relationship of these factors to the regulatory mechanisms responsible for the development and maintenance of the differentiated state. The experiments described here are designed to determine the relative role of both nuclear and extranuclear factors in these regulatory events. Initially, two teratocarcinoma-derived cell lines will be used for this study; a) an embryonal carcinoma cell line (247-Des or PCC48azal) which is multipotenial and tumorigenic and b) a parietal yolk sac cell line (PYS-2) which expresses various "differentiated" functions and is nontumorigenic. Somatic cell hybrids will be prepared between these cell lines, and characterized for their tumorigenic and differentiated state. Cells will then be fractionated into a) karyoplasts, or nuclei surrounded by a small rim of cytoplasm and plasma membrane and b) cytoplasts, or enucleated cells. Various types of recombinant cells will be prepared and examined for their differentiated and tumorigenic state. The regulatory role of extranuclear factors will be investigated in cybrids, or recombinant cells formed by the fusion of cytoplasts with whole cells. Similarly, the role of the nucleus will be investigated by preparation and characterization of binucleated cells, or cells formed by the fusion of karyoplasts and whole cells. Lastly, various reconstituted cells will be prepared by the fusion of isolated karyoplasts and cytoplasts. Characterization of recombinant cells formed by all possible crosses, should provide insight into a) the complexity of the regulatory network, b) the nuclear vs. extranuclear location of the various regulatory factors and c) the nature of the interaction between the regulatory factors controlling the differentiated and tumorigenic state of the cell.